Immunomodulation by small molecules can be achieved by identifying compounds that bind and activate Toll-Like Receptors (TLRs). TLRs play an important role in innate immune responses in mammals and are often the first line of defense against pathogens such as bacteria and viruses. The various TLRs vary in their abundance in different mammalian cell types and also vary regarding the molecular structures that bind the TLR and activate signaling pathways. These signaling pathways lead to the range of responses associates with innate immunity.
TLRs detect PAMPs (pathogen-associated molecular patterns) and stimulate immune cells via the MyD88-dependent interleukin 1 receptor (IL-1R)-TLR signaling pathway, which leads to activation of the transcription factor NF-κB2. Ten functional family members of TLRs (TLR1 to TLR10) have been identified in humans. Akira S. et al., Nature Immunol., 2, 675-680 (2001). TLR2, TLR4, and TLR5 are crucial for the recognition of peptidoglycan, lipopolysacharide, and flagellin. Hayashi, F. et al., Nature, 410, 1099-1103 (2001). TLR6 associates with TLR2 and recognizes lipoproteins from mycoplasma. Ozinsky, A., et al., Proc. Natl. Acad. Sci. USA., 97, 13766-13771 (2000). TLR9 detects bacterial DNA containing unmethylated CpG motifs and TLR3 activates immune cells in response to double-stranded RNA. Hemmi, H. et al., Nature, 408, 740-745 (2000).
A number of compounds, including guanosine analogs, substituted pyrimidines, and imidazoquinolines have been reported as ligands for TLR7. See, e.g., Hemmi et al., Nature Immunol., 3, 196-200 (2002) (imiquimod and R-848 (resiquimod)); Jurk et al., Nat. Immunol., 3, 499 (2002) (R-848); and Lee et al., Proc. Natl. Acad. Sci. USA, 100, 6646-6651 (2003) (wherein guanosine analogs loxoribine, 7-thia-8-oxoguanosine (isatoribine), and 7-deazaguanosine, and the imidazoquinolines imiquimod and R-848 (resiquimod) selectively activate TLR7).
Prior to being linked as potential TLR7 ligands, guanosine analogs and other D- and L-purine nucleosides have been the subject of considerable research the past two decades. See, e.g., Reitz et al., J. Med. Chem., 37, 3561-78 (1994); Michael et al., J. Med. Chem., 36, 3431-36 (1993) (immunomodulatory guanosine analogs having substituents at the 7- and/or 8-positions); U.S. Pat. No. 5,821,236 to Krenitsky et al. (disclosing 6-alkoxy derivatives of arabinofuranosyl purine derivatives that are useful for tumor therapy); U.S. Pat. No. 5,041,426 to Robins et al. (certain pyrimido[4,5-d]pyridimine nucleosides are disclosed in as being effective in treatment against L1210 in BDF1 mice); Revankar et al., J. Med. Chem., 27, 1489-96 (1984) β-Deazaguanine nucleosides and nucleotides demonstrating significant broad spectrum antiviral activity against certain DNA and RNA viruses);
A number of compounds known to be immunostimulants have recently been identified in the literature as TLR7 ligands, see, e.g., Heil et al., Eur. J. Immunol., 33(11), 2987-97 (2003), Lore et al., J. Immunol., 171(8), 4320-8 (2003), Nagase et al., J. Immunol., 171(8), 3977-82 (2003), Mohty et al., J. Immunol., 171(7), 3385-93 (2003), Pinhal-Enfield, et al., Am. J. Pathol., 163(2), 711-21 (2003), Doxsee et al, J. Immunol., 171(3), 1156-63 (2003), Bottcher et al., Neurosci. Lett., 344(1), 17-20 (2003), Kaisho et al., Curr. Mol. Med., 3(4), 373-85 (2003), Okada et al., Eur. J. Immunol., 33(4), 1012-9 (2003), Edwards et al., Eur. J. Immunol., 33(4), 827-33 (2003), Akira et al., Immunol. Lett., 85(2), 85-95 (2003), Ito et al., Hum. Immunol., 63(12), 1120-5 (2002), Rothenfusser et al., Hum. Immunol., 63(12), 1111-9 (2002), Yamamoto et al., J. Immunol., 169(12), 6668-72 (2002), Gibson et al., Cell Immunol., 218(1-2), 74-86 (2002), Horng et al., Nature, 420 (6913), 329-33 (2002), Yamamoto et al., Nature, 420(6913), 324-9 (2002), Applequist et al., Int. Immunol., 14(9), 1065-74 (2002), Sato et al., Int. Immunol., 14(7), 783-91 (2002); Jurk et al., Nat. Immunol., 3(6), 499 (2002); Hornung et al., J. Immunol., 168(9), 4531-7 (2002), Hemmi et al., Nat. Immunol., 3(2), 196-200 (2002); Bruno et al., Eur. J. Immunol., 31(11), 3403-12 (2001); Jarrossay et al., Eur. J. Immunol., 31(11), 3388-93 (2001); Miettinen et al., Genes Immun., 2(6), 349-55 (2001), Chuang et al., Eur. Cytokine Netw., 11(3), 372-8 (2000), and Du et al., Eur. Cytokine Netw., 11(3), 362-71 (2000).
These TLR7 ligands are known to stimulate immune responses in vitro and in animal species, and this has led to testing of the uses of these compounds for several therapeutic uses, including antiviral and cancer therapies. These compounds have been characterized as analogs or derivatives of a) guanosine, b) imidazoquinoline, and c) pyrimidine. See Akira, Current Opinion, 15, 5-11 (2003). One member (imiquimod) of the imidazoquinoline chemical class has been found effective for treating topical genital infections by papilloma virus. A second member of the imidazoquinoline class, resiquimod, has been tested for the treatment of HCV, but this compound failed to show anti-HCV effect at tolerated oral doses. Pockros et al., Gastroenterology, 124 (Suppl 1), A-766 (2003).
Thus, while there has been some limited use of TLR7 ligands for the treatment of immunological disease and viral infections; see, e.g. U.S. Pat. Nos. 5,041,426 and 4,880,784 to Robins et al. β-β-D-ribofuranosylthiazolo[4,5-d]pyridimines demonstrating significant immunoactivity, including murine spleen cell proliferation and in vivo activity against Semliki Forest virus); United States Patent Application Publication No. US 2003/0199461 and WO 03/045968 to Averett et al. β-β-D-ribofuranosylthiazolo[4,5-d]pyrimidine nucleosides demonstrating activity against acute and chronic infections of both RNA and DNA viruses); to date ligands have proved ineffective for the treatment or prevention of Hepatitis C virus.
It is also known that the oral administration of many purine nucleoside analogs are subject to difficulties arising from poor absorption, poor solubility, or degradation in the digestive tract as a result of acidic or alkaline conditions or the action of enzymes, and/or combinations of these phenomena. Thus there remains a need for purine nucleoside analogs with improved oral availability and administration that are used to modulate aspects of the immune system.
Moreover, immunomodulatory nucleosides have relatively poor oral tolerability when compared to that of the intravenous route. Also, the gastrointestinal tract presents a particular tolerability barrier to immunologic agents by virtue of the large amount of immune tissue associated with the intestinal wall (i.e., gut). Although this is an important biologic mechanism for preventing invasion of the body by gut flora, the immune tissue also may become preferentially affected after oral administration of immunomodulatory compounds because of the high local concentrations of the administered compound in the gut. This leads to undesirable side effects, for example in the case of immune activating agents there is observed gastroenteritis and localized hemorrhagic effects.
A solution to the problem of effective oral delivery of immunomodulators is not evident in the literature. Available evidence indicates that systemic levels of administered drugs in this class have been limited by gastrointestinal toxicities arising after low oral doses. Therefore there remains a need for immunomodulating TLR7 ligands that have improved oral availability and reduced gastrointestinal irritancy.